Cav-1 Protein Research Articles

The activated caveolin-3/μ-opioid receptor complex drives morphine-induced rescue therapy in failing hearts.

Opioid analgesics can alleviate ischaemia/reperfusion (I/R) injury in chronic heart failure. However, the underlying mechanisms and targets remain unknown. Here, we investigate if caveolin-3 (Cav3) interacts with μ opioid receptors and if Cav3-μ receptor interactions play a role in morphine-induced cardioprotection in failing hearts. Cav3 and μ receptor proteins in human and rat heart tissue were determined by western blot, immunofluorescence and co-immunoprecipitation. Methyl-β-cyclodextrin (MβCD), a destroyer of caveolae, and AAV-Cav3 shRNA were used to reduce Cav3 expression in failing rat hearts. CTOP, a specific μ antagonist, was administrated before morphine preconditioning in perfused failing heart models of myocardial I/R injury. Levels of Cav3 and μ receptor proteins were significantly higher in human and rat myocardial tissues with heart failure than in control tissues. Cav3 and μ receptor expression levels were positively correlated with disease severity. The signal of the cardiac Cav3 protein was colocalized with μ receptor in both the human and rat heart sections. Disruption of caveolae in the failing heart by either MβCD or AAV-Cav3 shRNA significantly inhibits morphine-induced phosphorylation of ERK1/2 and cardioprotection. Administration of CTOP substantially reduced Cav3 expression and morphine-induced cardioprotective effect in heart failure. Our data suggest that up-regulation of the Cav3/μ receptor complex is critical for morphine protection of the failing heart against I/R injury by regulating the ERK1/2 pathway. The activated Cav3/μ receptor complex is an understudied therapeutic target for opioid treatment of heart failure and ischaemic insult.

Caveolin-dependent endocytosis regulates arterial calcification

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB219 Introduction Patients with chronic kidney disease (CKD) have a significantly elevated cardiovascular risk, which is primarily due to premature vascular aging with arterial calcification - a substantial public health burden. Calcifying extracellular vesicles (EVs) are required for the onset and progression of calcification. Endocytosis and exocytosis are important for EV genesis and cargo loading, thus controlling EV release and propensities. Purpose We aim to investigate endocytosis, endosomal ripening, and exocytosis as critical mechanisms of EV-dependent mineralization. Methods and Results First, we characterized EVs from calcifying vascular smooth muscle cells (SMCs) compared to control SMCs. Western blot analysis revealed an increase in tissue nonspecific alkaline phosphatase (TNAP) among calcifying EVs but not in flotillin abundance. When compared to control EVs, calcifying EVs showed higher TNAP activity. Flow cytometry and a turbidity assay were then used to evaluate EV mineral cargo and aggregation potential. Compared to the control, EVs produced from SMCs cultivated under calcifying conditions had 5-fold more mineral-positive EVs (p=0.004) and a 3-fold higher nucleation potential (p<0.001). Next, endocytosis was blocked by targeting Dynamin-1 and –2, which inhibited caveolin- and clathrin-dependent endocytosis (Dynasore hydrate), or targeting clathrin-terminal domain, which inhibited solely clathrin-dependent endocytosis (Pitstop 2). Dynasore reduced matrix mineralization dose-dependently and lowered mineral-positive EVs by 18%, whereas Pitstop 2 had no effect, suggesting a role of caveolin-dependent endocytosis in EV-mediated calcification. Indeed, Cav-1 silencing inhibited matrix mineralization and Cav-1 protein was more abundant in calcifying EVs than in control EVs. Dynasore and Pitstop-2 did not affect the EV number measured by nanoparticle tracking analysis. Inhibition of endosomal ripening by Rab7 inhibition did not affect SMC calcification. Exocytosis was inhibited by Rab27a inhibition, which reduced mineral deposition (-32%, p=0.017) without affecting EV mineral or EV quantity. Conclusion EVs released from calcifying SMCs showed a higher abundance of mineral and TNAP proteins, suggesting a role in mineral transport and deposition. Our data suggest that caveolin-dependent endocytosis is essential in EV-mediated SMC calcification. Mineral cargo in EVs is independent of Rab7 and Rab27a pathways.

Motor unit loss and neuromuscular junction degeneration precede clinically diagnosed sarcopenia

Background: Degeneration of motoneuron and neuromuscular junctions (NMJ) and loss of motor units (MUs) contribute to age-related muscle wasting and weakness, leading to sarcopenia. However, these features have not been adequately investigated in humans. This study aimed to compare neuromuscular system integrity and function across different stages of sarcopenia, with a particular focus on NMJ stability and MU properties. We hypothesized that progressive impairment of the neuromuscular system would be a hallmark of the progression of sarcopenia. Methods: We recruited 42 healthy young individuals (Y) (aged 25.98±4.6 yr; 57% females) and 88 older individuals (aged 75.9±4.7 yr; 55% females). The older group underwent a sarcopenia screening according to the revised guidelines of the European Working Group on Sarcopenia in Older People (EWGSOP2). In all groups, knee extensor muscle force was evaluated by isometric dynamometry, muscle size by ultrasound and MUP properties by intramuscular electromyography (iEMG). MU number estimate (iMUNE) was also obtained. Blood samples and muscle biopsies were collected to assess biomarkers of neuromuscular degeneration. Results: 39 older individuals were non-sarcopenic (NS), 31 pre-sarcopenic (PS) and 18 sarcopenic (S). A progressive decline in quadriceps force, cross-sectional area and appendicular lean mass was detected with the different stages of sarcopenia. Handgrip force and short physical performance battery (SPPB) scores were also progressively reduced. iEMG recordings showed increased near fiber segment jitter in NS, PS and S compared to the Y group, highlighting a decreased NMJ transmission stability. Elevated concentration of serum C-terminal agrin fragment and altered Cav3 protein expression further supported the finding of age-related NMJ instability in all the older groups. The iMUNE was lower in all older groups, confirming age-related loss of MUs. Motoneuron firing rate was decreased in S vs Y. Age-related increases in MU potential complexity were also observed. These observations were accompanied by increased muscle denervation and axonal damage, respectively, indicated by the increase in NCAM-positive fibres and the increase in serum concentration of neurofilament light chain. Notably, most of these MU and NMJ parameters did not differ in older individuals with or without sarcopenia. Conclusion: Alterations in MU properties, axonal damage, altered innervation profile and NMJ instability are signatures of neuromuscular system ageing. These neuromuscular impairments are accompanied by muscle atrophy and weakness, however, they appear to precede clinically diagnosed sarcopenia, as they are already detectable in older NS individuals. These are important parameters to monitor in order to counteract these impairments and slow down the onset and progression of sarcopenia. The present work was funded by the PRIN project ‘NeuAge’ (2017CBF8NJ_001) to MVN and MAP and the Milky Way Foundation, the Baxter Foundation and the Li Ka Shing Foundation to HMB. EM was supported by the Stanford Wu Tsai Human Performance Institute fellowship and Marie Skłodowska-Curie Actions postdoctoral fellowship. We also acknowledge co-funding from Next Generation EU to MVN, in the context of the National Recovery and Resilience Plan, Investment PE8 — Project Age-It: “Ageing Well in an Ageing Society”. This resource was co-financed by the Next Generation EU [DM 1557 11.10.2022]. The views and opinions expressed are only those of the authors and do not necessarily reflect those of the European Union or the European Commission. Neither the European Union nor the European Commission can be held responsible for them. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cholesterol depletion induces mesenchymal properties in oral squamous cell carcinoma cell line.

Cholesterol in cell membranes is crucial for cell signaling, adhesion, and migration. Membranes feature cholesterol-rich caveolae with caveolin proteins, playing roles in epithelial-mesenchymal transition and cancer progression. Despite elevated cholesterol levels in tumors, its precise function and the effects of its depletion in oral squamous cell carcinoma remain unclear. The aim of this study was to evaluate the influence of cholesterol depletion in oral squamous cell carcinoma cell line and epithelial-mesenchymal transition process. Cholesterol depletion was induced on SCC-9 cells by methyl-β-cyclodextrin and cell viability, proliferation, apoptosis, and colony formation capacities were evaluated. Gene and protein expressions were evaluated by reverse transcription polymerase chain reaction (RT-qPCR) and Western Blot, respectively, and cell sublocalization was assessed by immunofluorescence. Cholesterol depletion resulted in alteration of oral squamous cell carcinoma cell morphology at different concentrations of methyl-β-cyclodextrin, as well as decreased cell proliferation and viability rates. Analysis of CAV1 transcript expression revealed increased gene expression in the treated SCC-9 during the 24 h period, at different concentrations of methyl-β-cyclodextrin: 5 , 7.5, 10, and 15 mM, in relation to parental SCC-9. CAV1 protein expression was increased, with subsequent dose-dependent decrease. A statistically significant difference was observed in samples treated with 5 mM of methyl-β-cyclodextrin (p = 0.02, Kruskal-Wallis test). The immunofluorescence assay showed lower cytoplasmic and membrane labeling intensity in the treated samples for CAV1. These findings indicate the modulation of cholesterol as a possible mechanism underlying the regulation of these molecules and activation of epithelial-mesenchymal transition in oral squamous cell carcinoma.

Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes by PKCα-associated signalling under cholesterol stimulation.

The secretion of bile salts transported by the bile salt export pump (BSEP) is the primary driving force for the generation of bile flow; thus, it is closely related to the formation of cholesterol stones. Caveolin-1 (Cav-1), an essential player in cell signalling and endocytosis, is known to co-localize with cholesterol-rich membrane domains. This study illustrates the role of Cav-1 and BSEP in cholesterol stone formation. Adult male C57BL/6 mice were used as an animal model. HepG2 cells were cultured under different cholesterol concentrations and BSEP, Cav-1, p-PKCα and Hax-1 expression levels were determined via Western blotting. Expression levels of BSEP and Cav-1 mRNA were detected using real-time PCR. Immunofluorescence and immunoprecipitation assays were performed to study BSEP and Hax-1 distribution. Finally, an ATPase activity assay was performed to detect BSEP transport activity under different cholesterol concentrations in cells. Under low-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels significantly increased, PKCα phosphorylation significantly decreased, BSEP binding capacity to Hax-1 weakened, and BSEP function increased. Under high-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels decreased, PKCα phosphorylation increased, BSEP binding capacity to Hax-1 rose, and BSEP function decreased. Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes via PKCα-associated signalling under cholesterol stimulation.

CAV1 Protein Encapsulated in Mouse BMSC-Derived Extracellular Vesicles Alleviates Myocardial Fibrosis Following Myocardial Infarction by Blocking the TGF-β1/SMAD2/c-JUN Axis.

Extracellular vesicles (EVs) derived from mouse bone marrow mesenchymal stem cells (mBMSCs) convey the CAV1 protein, influencing the TGF-β1/SMAD2/c-JUN pathway and thus the molecular mechanisms underlying myocardial fibrosis (MF) post-myocardial infarction (MI). Through various experimental methods, including transmission electron microscopy, Nanosight analysis, Western blot, ELISA, and qRT-PCR, we isolated, purified, and identified EVs originating from mBMSCs. Bioinformatics and experimental findings show a reduced expression of CAV1 in myocardial fibrosis tissue. Furthermore, our findings suggest that mBMSC-EVs can deliver CAV1 to cardiac fibroblasts (CFs) and that silencing CAV1 in mBMSC-EVs promotes CF fibrosis. In vivo studies further corroborated these findings. In conclusion, mBMSC-EVs mitigate myocardial fibrosis in MI mice by delivering the CAV1 protein, inhibiting the TGF-β1/SMAD2/c-JUN pathway.

Caveolin-1 is critical for hepatic iron storage capacity in the development of nonalcoholic fatty liver disease

BackgroundNonalcoholic fatty liver disease (NAFLD) is associated with disordered lipid and iron metabolism. Our previous study has substantiated the pivotal role of Caveolin-1 (Cav-1) in protecting hepatocytes and mediating iron metabolism in the liver. This study aimed to explore the specific mechanisms underlying the regulation of iron metabolism by Cav-1 in NAFLD.MethodsHepatocyte-specific Cav-1 overexpression mice and knockout mice were used in this study. Cav-1-knockdown of RAW264.7 cells and mouse primary hepatocytes were performed to verify the changes in vitro. Moreover, a high-fat diet and palmitic acid plus oleic acid treatment were utilized to construct a NAFLD model in vivo and in vitro, respectively, while a high-iron diet was used to construct an in vivo iron overload model. Besides, iron concentration, the expression of Cav-1 and iron metabolism-related proteins in liver tissue or serum were detected using iron assay kit, Prussian blue staining, Western blotting, immunofluorescence staining, immunohistochemical staining and ELISA. The related indicators of lipid metabolism and oxidative stress were evaluated by the corresponding reagent kit and staining.ResultsSignificant disorder of lipid and iron metabolism occurred in NAFLD. The expression of Cav-1 was decreased in NAFLD hepatocytes (P < 0.05), accompanied by iron metabolism disorder. Cav-1 enhanced the iron storage capacity of hepatocytes by activating the ferritin light chain/ferritin heavy chain pathway in NAFLD, subsequently alleviating the oxidative stress induced by excess ferrous ions in the liver. Further, CD68+CD163+ macrophages expressing Cav-1 were found to accelerate iron accumulation in the liver, which was contrary to the effect of Cav-1 in hepatocytes. Positive correlations were also observed between the serum Cav-1 concentration and the serum iron-related protein levels in NAFLD patients and healthy volunteers (P < 0.05).ConclusionsThese findings confirm that Cav-1 is an essential target protein that regulates iron and lipid metabolic homeostasis. It is a pivotal molecule for predicting and protecting against the development of NAFLD.

PM2.5 triggers autophagic degradation of Caveolin-1 via endoplasmic reticulum stress (ERS) to enhance the TGF-β1/Smad3 axis promoting pulmonary fibrosis

Air pollution is highly associated with respiratory diseases. However, the influence and mechanism of particulate matter with aerodynamic equal to or less than 2.5 μm (PM2.5) in lung homeostasis remain unclear. Herein, we demonstrated the induction of pulmonary fibrosis (PF) by PM2.5 exposure. The animal model showed that PM2.5 exposure could activate the oxidative stress and inflammation response, promoting epithelial-mesenchymal transition and accumulation of collagen, high expression of pro-fibrotic factors, and pathological characteristics of fibrosis. The proteomic analysis indicated that PM2.5 exposure decreased the expression of caveolin-1 (Cav-1), and many differential proteins were enriched in the TGF-β1/Smad, endoplasmic reticulum stress (ERS) and autophagy pathways. Combining in vivo and in vitro experiments, it was found that PM2.5 exposure could reduce Cav-1 protein levels and activate TGF-β1/Smad3 signaling pathways through ERS and autophagy pathways, thereby inducing cell apoptosis and promoting pulmonary fibrosis. However, inhibiting ERS could alleviate the occurrence of autophagy, and blocking the autophagy system could increase the level of Cav-1 protein and inhibit TGF- β 1/Smad3 signaling pathway to improve pulmonary fibrosis. Therefore, we demonstrated that the exposure of PM2.5 could enhance the ERS induced-autophagy-mediated Cav-1 degradation, thus activating the TGF-β1/Smad3 axis to promote pneumonocytes apoptosis and overproduction of extracellular matrix (ECM), finally aggravating PF. Moreover, our findings revealed that intermittent exposure to high doses of PM2.5 was more toxic than continuous exposure to low dose.

Docetaxel radiosensitizes castration-resistant prostate cancer by downregulating CAV-1

Purpose: Docetaxel (DXL), a noted radiosensitizer, is one of the few chemotherapy drugs approved for castration-resistant prostate cancer (CRPC), though only a fraction of CRPCs respond to it. CAV-1, a critical regulator of radioresistance, has been known to modulate DXL and radiation effects. Combining DXL with radiotherapy may create a synergistic anticancer effect through CAV-1 and improve CRPC patients’ response to therapy. Here, we investigate the effectiveness and molecular characteristics of DXL and radiation combination therapy in vitro. Materials and methods: We used live/dead assays to determine the IC50 of DXL for PC3, DU-145, and TRAMP-C1 cells. Colony formation assay was used to determine the radioresponse of the same cells treated with radiation with/without IC50 DXL (4, 8, and 12 Gy). We performed gene expression analysis on public transcriptomic data collected from human-derived prostate cancer cell lines (C4-2, PC3, DU-145, and LNCaP) treated with DXL for 8, 16, and 72 hours. Cell cycle arrest and protein expression were assessed using flow cytometry and western blot, respectively. Results: Compared to radiation alone, combination therapy with DXL significantly increased CRPC death in PC3 (1.48-fold, p < .0001), DU-145 (1.64-fold, p < .05), and TRAMP-C1 (1.13-fold, p < .05) at 4 Gy of radiation. Gene expression of CRPC treated with DXL revealed downregulated genes related to cell cycle regulation and upregulated genes related to immune activation and oxidative stress. Confirming the results, G2/M cell cycle arrest was significantly increased after treatment with DXL and radiation. CAV-1 protein expression was decreased after DXL treatment in a dose-dependent manner; furthermore, CAV-1 copy number was strongly associated with poor response to therapy in CRPC patients. Conclusions: Our results suggest that DXL sensitizes CRPC cells to radiation by downregulating CAV-1. DXL + radiation combination therapy may be effective at treating CRPC, especially subtypes associated with high CAV-1 expression, and should be studied further.

Caveolin-3 loss linked with the P104L LGMD-1C mutation modulates skeletal muscle mTORC1 signalling and cholesterol homeostasis.

Caveolins are the principal structural components of plasma membrane caveolae. Dominant pathogenic mutations in the muscle-specific caveolin-3 (Cav3) gene isoform, such as the limb girdle muscular dystrophy type 1C (LGMD-1C) P104L mutation, result in dramatic loss of the Cav3 protein and pathophysiological muscle weakness/wasting. We hypothesize that such muscle degeneration may be linked to disturbances in signalling events that impact protein turnover. Herein, we report studies assessing the effects of Cav3 deficiency on mammalian or mechanistic target of rapamycin complex 1 (mTORC1) signalling in skeletal muscle cells. L6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 was abolished by CRISPR/Cas9 genome editing (Cav3 knockout [Cav3KO]) prior to performing subcellular fractionation and immunoblotting, analysis of real-time mitochondrial respiration or fixed cell immunocytochemistry. Skeletal muscle from wild-type and Cav3-/- mice was processed for immunoblot analysis of downstream mTORC1 substrate phosphorylation. Cav3 was detected in lysosomal-enriched membranes isolated from L6 myoblasts and observed by confocal microscopy to co-localize with lysosomal-specific markers. Cav3P104L expression, which results in significant (~95%) loss of native Cav3, or CRISPR/Cas9-mediated Cav3KO, reduced amino acid-dependent mTORC1 activation. The decline in mTORC1-directed signalling was detected by immunoblot analysis of L6 muscle cells and gastrocnemius Cav3-/- mouse muscle as judged by reduced phosphorylation of mTORC1 substrates that play key roles in the initiation of protein synthesis (4EBP1S65 and S6K1T389 ). S6K1T389 and 4EBP1S65 phosphorylation reduced by over 75% and 80% in Cav3KO muscle cells and by over 90% and 30% in Cav3-/- mouse skeletal muscle, respectively. The reduction in protein synthetic capacity in L6 muscle cells was confirmed by analysis of puromycylated peptides using the SUnSET assay. Cav3 loss was also associated with a 26% increase in lysosomal cholesterol, and pharmacological manipulation of lysosomal cholesterol was effective in replicating the reduction in mTORC1 activity observed in Cav3KO cells. Notably, re-expression of Cav3 in Cav3KO myoblasts normalized lysosomal cholesterol content, which coincided with a recovery in protein translation and an associated increase in mTORC1-directed phosphorylation of downstream targets. Our findings indicate that Cav3 can localize on lysosomal membranes and is a novel regulator of mTORC1 signalling in muscle. Cav3 deficiency associated with the Cav3P104L mutation impairs mTORC1 activation and protein synthetic capacity in skeletal muscle cells, which may be linked to disturbances in lysosomal cholesterol trafficking and contribute to the pathology of LGMD-1C.

Caveolin-1 depletion attenuates hepatic fibrosis via promoting SQSTM1-mediated PFKL degradation in HSCs.

The key glycolytic enzyme phosphofructokinase (PFK) is responsible for maintaining glycolytic stability and an important energy source for activating hepatic stellate cells (HSCs). However, its regulation in activated HSCs remains unclear. Caveolin-1 (Cav1), a major constituent of caveolae, has emerged as a key target for triggering glycolysis. However, the relationship between Cav1 and glycolysis during HSC activation is not well established. In this study, Cav1 was upregulated in mouse and human fibrotic liver tissues. We concluded that HSC-specific Cav1 knockdown markedly alleviates liver injury and fibrosis. Mechanistically, Cav1 was elevated during primary mouse HSC activation, competing with SQSTM1 for the regulatory subunit of PFK liver type and inhibiting the SQSTM1-mediated autophagy-independent lysosomal degradation pathway to sustain HSC activation. We also identified the heptapeptide alamandine as a promising therapeutic agent that downregulates Cav1 protein levels via proteasomal degradation and may impair glycolysis. Our study provides evidence of the crucial role and mechanism of Cav1 in the glucose metabolic network in HSCs and highlights Cav1 as a critical therapeutic target for the treatment of liver fibrosis.

Silencing information regulator 1 ameliorates lipopolysaccharide-induced acute lung injury in rats via the upregulation of caveolin-1

BackgroundAcute lung injury (ALI) is an intractable medical problem linked with to high morbidity and mortality all over the worldglobally. The prognosis of advanced acute lung injury remains persistently poor due to its underlying mechanisms remain unclear.Despite advancements in medical research, the its prognosis of advanced ALI remains persistently poor due to unclear underlying mechanisms. We aimed to investigate the protective effects of silencing information regulator 1 (SIRT1) on lipopolysaccharide (LPS)-induced acute lung injuryALI and to reveal its underlying molecular mechanism. MethodsMale Sprague-–Dawley rats were divided grouped into 4 groupsfour: normal saline group (group NS), lipopolysaccharide group (group L), SIRT1 activator SRT1720-pretreated group (group S), and SIRT1 inhibitor EX527- pretreated group (group E). Rats They were intranasally dripped with LPS to establish the model of ALI modelsacute lung injury respectively. We investigated the effect of SIRT1 on acute lung injury by analysing We analyzed the CT images of the rat lungs and used, HE staining, lung wet-to-dry ratio, inflammatory factor expression, lung injury marker expression, immunohistochemistry, and related mRNA expression to determine the effect of SIRT1 on ALI. ResultsOur results show that LPS induction produced resulted in acute lung injury, ALI and disrupting disrupted normal SIRT1 expression, which led to the overexpression of STAT3, TLR4, TNF-ɑ, and IL-6 and suppression of Cav-1 expression. Upregulation The upregulation of Cav-1 protein and mRNA following the administration of an SIRT1 agonist resulted in reduced lung injury. SRT1720 pretreatment was closely associated with reduced expressions of STAT3,TLR4, TNF-ɑ, and IL-6. ALI lung injury was more severeworsened after administration of SIRT1 inhibitors, and the changes in the above indicators were reversed. ConclusionsThese results suggest that SIRT1 may protect against LPS-induced acute lung injuryALI via by counteracting inflammatory remissionion, and this protective effect might may be mediated by suppressing STAT3 to activate the expression ofinduce Cav-1 expression.

Puerarin Attenuates High-Glucose and High-Lipid-Induced Inflammatory Injury in H9c2 Cardiomyocytes via CAV3 Protein Upregulation.

Inflammation plays a crucial role in the development of diabetic cardiomyopathy (DCM), including inflammation caused by high-glucose and high-lipid (HGHL). Targeting inflammation may provide a useful strategy for preventing and treating DCM. Puerarin has been shown to reduce the inflammation, apoptosis and hypertrophy of cardiomyocytes induced by HGHL, in which this study aims to investigate the underlying mechanisms. H9c2 cardiomyocytes cultured with HGHL were used to establish a cell model of DCM. Puerarin was then placed to these cells for 24 hours. The effects of HGHL and puerarin on cell viability and apoptosis were investigated by the Cell Proliferation, Toxicity Assay Kit (CCK-8) and flow cytometry. Morphological changes of cardiomyocytes was observed by HE staining. CAV3 proteins in H9c2 cardiomyocytes were altered by transient transfection of CAV3 siRNA. IL-6 was detected by ELISA. The Western blot was performed to determine the CAV3, Bcl-2, Bax, pro-Caspase-3, cleaved-Caspase-3, NF-κB (p65) and p38MAPK proteins. Puerarin treatment reversed the cells viability, hypertrophy in morphology, inflammation (showed by p-p38 and p-p65 and IL-6) and apoptosis-related damage (showed by cleaved-Caspase-3/pro-Caspase-3/Bax, Bcl-2 and flow cytometry) of the H9c2 cardiomyocyte caused by HGHL. Puerarin treatment also restored the decrease of CAV3 proteins of the H9c2 cardiomyocyte caused by HGHL. When silenced the expression of CAV3 proteins with SiRNA, puerarin failed to decreased p-p38 and p-p65 and IL-6, and could not reversed cell viability and morphological damage. In contrast to the simple CAV3 silenced group, the CAV3 silenced with NF-κB pathway or p38MAPK pathway inhibitors, significantly downregulated the p-p38, p-p65 and IL-6. Puerarin upregulated CAV3 protein expression in H9c2 cardiomyocytes and inhibited the NF-κB and p38MAPK pathways, thereby reducing HGHL-induced inflammation and may related to the apoptosis and hypertrophy of cardiomyocytes.

Four Looks to a Gene (P1-8.013)

<h3>Objective:</h3> We present a family with varied clinical presentations of a genetically confirmed muscular dystrophy. <h3>Background:</h3> A 41-year-old male presented with several years of muscle fatigue, pain and twitching across his chest and proximal muscles. He was diagnosed with neuronal hyperexcitability. Exam showed normal muscle strength, but there were visible rippling in proximal muscles. Cardiac workup was unrevealing. EMG showed nonspecific myopathic features and CPK was in the 400’s. His 9-year-old son has been diagnosed with muscular dystrophy and two daughters has elevated CPKs. Our patient was genetically confirmed to have a heterozygous C.80G&gt;A(p.Arg27Gln) mutation in the Caveolin-3 gene via Invitae muscular dystrophy testing. This missense mutation causes an amino acid change from Arg to Gln in the Caveolin 3 protein, which leads to a decrease of Cav-3 protein expression in skeletal muscle tissue. It has an autosomal dominant inheritance pattern. <h3>Design/Methods:</h3> Not applicable <h3>Results:</h3> Mutations in the Caveolin-3 gene have been described in a broad spectrum of clinical presentations including limb girdle muscular dystrophy, rippling muscle disease, idiopathic persistent hyperCKemia, and distal myopathy. Muscular dystrophy can present with a wide variety of phenotypes and there is even variation within the same gene mutations, as is the case in caveolin 3 mutations. This may lead to delay in diagnosis and supportive treatments that a family may require. The Invitae muscular dystrophy panel can be helpful in expediting diagnosis for multiple family members. It is also important to remember that muscular dystrophy can mimic muscle hyperexcitability syndrome, as was the case in our patient with rippling muscle disease. <h3>Conclusions:</h3> Muscular dystrophy can present with distinct phenotypic variations. A good family history and awareness of clinical symptoms can be helpful for diagnosis. CAV3 mutations cause LGMD1C which can present with at least four different phenotypes. <b>Disclosure:</b> Dr. Rahimi has nothing to disclose. Dr. Memar Ardestani has nothing to disclose. Dr. Prabhu has nothing to disclose. Dr. Undavia has nothing to disclose.

Cardioprotective and anti-inflammatory effects of Caveolin 1 in experimental diabetic cardiomyopathy.

Previous studies of the Caveolin 1 (Cav1) protein and caveolae, which are lipid raft structures found on the plasma membranes of certain cells, are associated with fat metabolism disorders, inflammation, diabetes, and cardiovascular disease. However, there have been no reports linking Cav1 to diabetic cardiomyopathy (DCM). In the present study, we established a relationship between Cav1 and the development of DCM. We found that compared with Cav1+/+ mice, Cav1-/- diabetic mice exhibited more severe cardiac injury, increased activation of NF-κB signaling, and up-regulation of downstream genes, including hypertrophic factors and inflammatory fibrosis factors in heart tissues. Additionally, in vitro results showed that knocking down Cav1 further activated HG-induced NF-κB signaling, increased the expression of downstream target genes, and decreased the expression of inhibitor α of NF-κB (iκBα), all of which have been linked to DCM pathogenesis. In contrast, Cav1 overexpression resulted in the opposite effects. Our study suggests that Cav1 knockdown promotes cardiac injury in DCM by activating the NF-κB signaling pathway, and targeting Cav1 may lead to the development of novel treatments for DCM.

Reduced endothelial caveolin-1 underlies deficits in brain insulin signalling in type 2 diabetes.

Patients with type 2 diabetes exhibit severe impairments in insulin signalling in the brain and are five times more likely to develop Alzheimer's disease. However, what leads to these impairments is not fully understood. Here, we show reduced expression of endothelial cell caveolin-1 (Cav-1) in the db/db (Leprdb) mouse model of type 2 diabetes. This reduction correlated with alterations in insulin receptor expression and signalling in brain microvessels as well as brain parenchyma. These findings were recapitulated in the brains of endothelial cell-specific Cav-1 knock-out (Tie2Cre; Cav-1fl/fl) mice. Lack of Cav-1 in endothelial cells led to reduced response to insulin as well as reduced insulin uptake. Furthermore, we observed that Cav-1 was necessary for the stabilization of insulin receptors in lipid rafts. Interactome analysis revealed that insulin receptor interacts with Cav-1 and caveolae-associated proteins, insulin-degrading enzyme and the tight junction protein Zonula Occludence-1 in brain endothelial cells. Restoration of Cav-1 in Cav-1 knock-out brain endothelial cells rescued insulin receptor expression and localization. Overall, these results suggest that Cav-1 regulates insulin signalling and uptake by brain endothelial cells by modulating IR-α and IR-β localization and function in lipid rafts. Furthermore, depletion of endothelial cell-specific Cav-1 and the resulting impairment in insulin transport leads to alteration in insulin signalling in the brain parenchyma of type 2 diabetics.

A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome.

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease.

Antitumor Activity of Taxol Engross Taxol-Caveolin-1 Interaction via Lipid Raft Structure-"Caveolae".

Taxol is one of the most widely used natural antitumor drugs that have shown considerable success in treating cancers of different lineage. However, the development of resistance to taxol is still a significant issue. Caveolae, the cave-like structures found on the surface of many cancerous cells, are enriched in cholesterol and are known to play a pivotal role in drug uptake. Caveolin-1 (Cav-1), the principal structural proteins of the caveolae, interacts with signaling molecules through a scaffolding domain. In the present study, we observed that Cav-1-GFP clusters were instantly recruited to the cell membrane. Interestingly, Caveolae formation followed by internalization was observed after the treatment with time. The recruitment and the formation of the Cav-1-GFP clusters are provided in supplementary video 2 (SV2). The results obtained from molecular docking indicate favorable taxol-Cav-1 interaction. To further confirm the influence of Cav-1 proteins in the uptake and effects of taxol, the cells were treated with beta-cyclodextrin (β-CD), cholesterol, and taxol combinations. The result suggests that the depletion of cholesterol in HeLa cells makes them less susceptible to taxol at a lower concentration. These observations provide evidence of the interaction between Cav-1 and taxol. Further studies that may elucidate the molecular mechanism of uptake of taxol through caveolae/Cav-1 will help to determine if Cav-1 can be used to increase the uptake of taxol by cancer cells and sensitize the drug-resistant cancer cells to taxol.

Expression of a full-length influenza virus hemagglutinin in Escherichia coli

Fusion or co-expression with other proteins is an effective option for improving recombinant protein expression in prokaryotic hosts. In this study, recombinant Escherichia coli strains expressing full-length influenza hemagglutinin (HA) were constructed to test the effect of co-expression of Cav1 protein or fusion with Oct1 upon expression of soluble HA. While HA alone was not expressed, a large amount of HA expression was observed upon co-expression with Cav1, which forms heterologous caveolae in the cytosol. When the DNA-binding protein Oct1 was used as a fusion partner, the HA solubility was improved. This study demonstrates a novel approach to achieve soluble expression of HA in E. coli.

Caveolin-1 inhibition mediates the opposing effects of alcohol on γ-secretase activity in arterial endothelial and smooth muscle cells.

Notch is important to vessel homeostasis. We investigated the mechanistic role of caveolin-1 (Cav-1) in mediating the effects of alcohol (Ethanol/EtOH) on the γ-secretase proteolytic activity necessary for Notch signaling in vascular cells. Human coronary artery endothelial cells (HCAEC) were treated with EtOH (0-50 mM), Notch ligand delta-like ligand 4 (Dll4), and the γ-secretase inhibitor DAPT. EtOH stimulated Notch signaling in HCAEC as evidenced by increased Notch receptor (N1, N4) and target gene (hrt2, hrt3) mRNA levels with the most robust response achieved at 25 mM EtOH. Ethanol (25 mM) stimulated γ-secretase proteolytic activity, to the same extent as Dll4, in HCAEC membranes. Ethanol inhibited Cav-1 mRNA and protein levels in HCAEC. Caveolin-1 negatively regulated γ-secretase activity in HCAEC as Cav-1 knockdown stimulated it, while Cav-1 overexpression inhibited it. Moreover, Cav-1 overexpression blocked the stimulatory effect of EtOH on γ-secretase activity in HCAEC. Although EtOH also inhibited Cav-1 expression in human coronary artery smooth muscle cells (HCASMC), EtOH inhibited γ-secretase activity in HCASMC in contrast to its effect in HCAEC. The inhibitory effect of EtOH on γ-secretase in HCASMC was mimicked by Cav-1 knockdown and prevented by Cav-1 overexpression, suggesting that in these cells Cav-1 positively regulates γ-secretase activity. In conclusion, EtOH differentially regulates γ-secretase activity in arterial EC and SMC, being stimulatory and inhibitory, respectively. These effects are both mediated by caveolin-1 inhibition which itself has opposite effects on γ-secretase in the two cell types. This mechanism may underlie, in part, the effects of moderate drinking on atherosclerosis.